Process using catalyst comprising soluble polymer and polymino acid

ABSTRACT

There is provided a process for the addition of a nucleophile across an electron poor carbon-carbon double bond (a Michael addition) comprising contacting in a solvent: i) a nucleophile; ii) a compound comprising an electron poor double bond; and iii) a catalyst comprising a soluble polymer and a polyamino acid.

This application is a 371 of PCT/GB01/02465, filed Jun. 1, 2001.

The present invention relates to a catalyst. In particular the presentinvention relates to a catalyst comprising a polyamino acid which may beutilised in a Michael addition reaction.

The ability of poly-amino adds to catalyse Michael additions such asasymmetric epoxidation¹ was discovered by Juliá and Colonna in 1980².Developments to the chemistry by Roberts^(3,4) have broadened the rangeof substrates, and the methodology has been applied to a range ofsynthetic targets^(5,6).

Prior art system have relied on heterogeneous system wherein thepolyamino acid is free or is bound to a solid support.

Tetrahedron Letters, Vol. 39, 1998, 9297-9300 describes an insoluble,heterogeneous catalyst involving the attachment of polyamino acids topolystyrene beads via a PEG-spacer.

Tetrahedron: Asymmetry Vol. 9, 1998 by Pu (pp 1457-1477) similarlydetails asymmetric epoxidations catalysed by insoluble, heterogeneouspolyamino acid catalysts.

A review by Gravert and Janda in Chem. Rev. Vol. 97, 1997 (pp 489-509)describes a variety of soluble polymer supports and details (p 495)oligoalanine and oligovaline units linked to glycine-PEG. The linkage ofthe polyamino acid to PEG Is an ester linkage. The ester linkage wouldbe unsuitable for Michael addition reactions involving basic hydrogenperoxide. Moreover the Review by Gravert and Janda and the initialpapers by Bonora et al Gazz Chim Ital., 1980, 503 and Makromol Chem.,1979, 2095 do not teach the reader how to accomplish Michael additionreactions such as asymmetric epoxidation reactions. No mention is madeof any catalytic function of the PEG-o-Gly-polyamino acids.

The prior art systems which have relied on heterogeneous system exhibita number of disadvantages. These systems provide low enantiomericexcess, particularly in systems comprising short chain (<10) amino acidsFurthermore, the heterogeneous nature of such system may limit theirindustrial applicability for reasons of ease of handling, slow reactionrates and limited substrates. Moreover heterogeneous systems are notreadily characterised by IR and NMR techniques.

The present invention alleviates the problems of the prior art.

In one aspect the present invention provides a process for the additionof a nucleophile across an electron poor carbon-carbon double bond (aMichael addition) comprising contacting in a solvent (i) a nucleophile;(ii) a compound comprising an electron poor double bond; and (iii) acatalyst comprising a soluble polymer (SSL) and a polyamino acid (PAA).

It will be appreciated by one skilled in the art that by the term“Michael addition” it is meant an addition of a nucleophile across adouble bond conjugated with an electron withdrawing group. Theepoxidation of an enone, a typical Michael addition, is exemplifiedbelow:

In the present specification by the term “electron poor” it is meant agroup having an electron withdrawing group on at least one terminal atomof the double bond.

In the present specification by the term “polyamino acid” it is meant acompound comprising two or more linked amino acid units. Such compoundscomprising a relatively low number of amino acid units may sometimes bereferred to as oligoamino acids.

The following abbreviations are used in the present specification

-   Amino-PEG: polyoxyethylene (bis) amine [purchased from Sigma, made    from PG of MW 3350]-   AR: analytical reagent-   Boc: tert-butoxycarbonyl-   DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene-   DOM: dichloromethane [was distilled from calcium hydride]-   DIC: 1,3-diisopropylcarbodiimide-   DMF: dimethylformamide [was purchased dry from Avocado and stored    under N₂]-   ee: enantiomeric excess-   Fmoc: 9-fluorenylmethoxycarbonyl-   HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexaflurophosphate-   HPLC: high performance liquid chromatography-   Leu-NCA: L-leucine N-carboxyanhydride-   M-PEG: polyethylene glycol monomethyly ether [various MW products    purchased from Fluka]-   PEG: polyethylene glycol [polyoxyethylene]-   PLL: poly-l-leucine-   UHP: Urea-hydrogen peroxide complex

It has been found that the catalysts of the present invention offer anumber of advantages. For embodiments of the present invention theseadvantages include:

-   -   for a given chain length of PAA, Increased catalytic activity        may be observed. Less catalyst is therefore required for a given        activity and the catalyst cost is reduced    -   the provision of a solubilised catalyst allows for catalysis in        a homogeneous system. Homogeneous systems provide        -   increased reaction speeds        -   increase in the number of possible substrates        -   increased degree of selectivity

With regard to the high degree of selectivity it has been identifiedthat an enantiomeric excess far in excess of that of the prior art maybe achieved by the process of the present invention. The enantiomericexcess is calculated using the following formula${\mathbb{e}\mathbb{e}} = {\frac{\left( {A - B} \right)}{\left( {A + B} \right)} \times 100}$wherein A is the amount of desired enantiomer and B is the amount ofundesired enantiomer.

The homogeneous of the present invention allows for the operation of theprocess in a continuous manner. The homogeneous catalyst system may beintroduced into a membrane reactor comprising a semi-permeable membranewherein the membrane is impermeable to the substrate and catalyst andpermeable to the reaction product. The catalyst is retained in thereactor. Substrate may be fed to the reactor and on conversion it maypass across the membrane and out the reactor. Such continuous operationis not possible with the heterogeneous systems of the prior art becausethe heterogeneous catalyst will block the membrane.

Nucleophile

Preferably the nucleophile of the present process is selected fromoxygen and sulphur. More preferably the nucleophile is oxygen.

When the nucleophile is oxygen, the oxygen nucleophile is preferablyprovided by a peroxide group.

When the nucleophile is sulphur, the sulphur nucleophile may be providedby a ⁻SPh group.

Compound Having Electron Poor Double Bond

Preferably the double bond is a carbon-carbon double bond.

As disclosed above an electron withdrawing group is attached to aterminal atom of the double bond of (ii). The electron withdrawing groupmay be selected from carbonyl, —CN and —NO₂.

In a preferred aspect the double bond is a carbon-carbon double bond andthe electron withdrawing group is a carbonyl group. In a particularlypreferred aspect the electron withdrawing group and the carbon-carbondouble bond comprise an enone group. In a highly preferred aspect thecompound comprising the electron poor double bond is a compound of thestructure

Catalyst

In preferred aspects of the present invention the catalyst is of theformula or comprises a group of the formula SSL-linker-PAA, wherein“linker” is a linker bond or an optional linker group.

Further preferred catalysts include those of the formulaX-SSL-linker-PAA-Y, or X-PAA-linker-SSL-linker-PAA-Y, wherein X and Yare independently selected from OMe and NH₂, and “linker” is a linkerbond or an optional linker group.

In each of the aspects above preferably Y is NH₂.

Linker

It is preferred that the linker is non-cleavable under the reactionconditions of the present process. It has been identified that apreferred non-cleavable linker bond is an amide bond.

The SSL-linker-PAA catalyst is typically prepared from SSL-(linkinggroup) and PAA. In this aspect the linking group is or comprises anucleophilic group. The nucleophilic linking group with attack the PAAto provide the corresponding linker group. Preferably the linking groupis or comprises a —NH₂ group. More preferably the linking group isselected from —O-hydrocarbyl-NH₂, -hydrocarbyl-NH₂, —C(CH₂NH₂)₂(NH₂).

For linking groups such as —C(CH₂NH₂)₂(NH₂).more than one linking moietyis provided for attachment to a PAA. In these aspects the catalyst maybe of the formula or comprise a group of the formula SSL-linker-(PAA)n,wherein n is an integer greater than 1.

The term “hydrocarbyl group” as used herein means a group comprising atleast C and H and may optionally comprise one or more other suitablesubstituents. Examples of such substituents may include halo, alkoxy,nitro, an alkyl group, a cyclic group etc. In addition to thepossibility of the substituents being a cyclic group, a combination ofsubstituents may form a cyclic group. If the hydrocarbyl group comprisesmore than one C then those carbons need not necessarily be linked toeach other. For example, at least two of the carbons may be linked via asuitable element or group. Thus, the hydrocarbyl group may containhetero atoms. Suitable hetero atoms will be apparent to those skilled inthe art and include, for instance, sulphur, nitrogen and oxygen. Anon-limiting example of a hydrocarbyl group is an acyl group.

A typical hydrocarbyl group is a hydrocarbon group. Here the term“hydrocarbon” means any one of an alkyl group, an alkenyl group, analkynyl group, which groups may be linear, branched or cyclic, or anaryl group. The term hydrocarbon also includes those groups but whereinthey have been optionally substituted. If the hydrocarbon is a branchedstructure having substituent(s) thereon, then the substitution may be oneither the hydrocarbon backbone or on the branch; alternatively thesubstitutions may be on the hydrocarbon backbone and on the branch.

Preferably the hydrocarbon of the linking group is C₁₋₆ alkyl, morepreferably (CH₂)₁₋₆.

The PAA may be attached to the linking group by formation of the PAA andsubsequent connection to the linking group, or may be synthesised bystepwise addition of amino acid units to the linking group.

PAA

The polyamino acid may be a homopolymer of one amino acid selected from,or a copolymer consisting of two or more amino acids selected from,cysteine, glycine, neopentyiglycine, alanine, valine, leucine,norleucine, phenylalanine, tyrosine, serine, cystine, threonine,methionine, di-iodotyrosine, thyroxine, dibromotyrosine, tryptophan,proline, hydroxyproline, aspartic acid, glutamic acid, β-hydroxyglutamicacid, omithine, arginine, lysine and histidine.

Preferably the polyamino acid is a homopolymer of one amino acidselected from, or a copolymer consisting of two or more amino acidsselected from, leucine, alanine and neopentylglycine. In a highlypreferred aspect the polyamino acid is a homopolymer of leucine.

Thus in a further aspect the present invention provides a catalystcomprising a soluble polymer and a polyamino acid, wherein the polyaminoacid is a homopolymer of an amino acid selected from, or a copolymerconsisting of two or more amino acids selected from, leucine, alanineand neopentylglycine.

The PAA preferably contains from 2 to 50, preferably 5 to 25, morepreferably 5 to 15, for example 5, 10 or 15 amino acid residues.

SSL

The soluble polymer is selected such that the catalyst is soluble in thesolvent in which the present process is conducted. The soluble polymermay be a homopolymer or a copolymer of monomers selected from styrene,vinyl alcohol, ethylene imine, acrylic acid, methylene oxide, ethyleneglycol, propylene oxide and acrylamide.

In a preferred aspect the soluble polymer is polyethylene glycol (PEG)or a derivative thereof. Derivatives include polyethylene glycolmonomethyl ether (M-PEG) and polyethylene glycol(bis)amine [amino-PEG].

The SSL or the solvent system of the present process may be selectedsuch that the catalyst may be easily recovered after use in the presentprocess. A SSL may be chosen which is soluble in one solvent and yetinsoluble in a second solvent. After reaction the catalyst may beextracted into the second solvent. The insolubility of the SSL in thissolvent will result in release of the catalyst from solution. Thecatalyst may then be readily collected, for example by filtration.

EXAMPLES

In the present Examples soluble catalysts are constructed by initiatingthe polymerisation of L-leucine N-carboxyanhydride with amino-PEG ofaverage MW 3500 (above). This amine is preferred to underivatized PEG asan amide linkage to the growing polypeptide is desirable in order forthe catalyst to be stable under the basic conditions utilised forepoxidation. It has been shown that the average chain length requiredfor a good asymmetric catalyst has been reduced by the use of solublecatalysts. Using soluble catalysts with an average chain length as lowas 5, the test substrate chalcone may be epoxidized (below) with an eeof 97%; far greater than for using an oligoleucine 5 mer supported on aninsoluble resin. Exact numbers of leucine residues have been coupled tothe support using solution phase peptide synthesis to investigate thisfurther.

10 mer L-leucine N-carboxyanhydride with amino-PEG was used in the abovecatalysis and compared to the catalysis provided by an analogous 10 merheterogeneous amino acid of the prior art. The HPLC traces of thereaction products are shown as FIGS. 1 and 2. The highlighted peaks are

It can be clearly seen from these traces that the catalyst of thepresent invention provides both a high degree of conversion and a highee when compared to the heterogeneous system of the prior art.

Example 1 Synthesis of Polyleucine on Polyethylene Glycol MonomethylEther (M-PEG)

Although some respectable ee s were observed for catalysts of this type,two main problems hampered this approach. Firstly, the difficulty ofobtaining a dry enough sample of M-PEG to ensure that this, rather thanany water present, initiated the polymerisation of the leu-NCA Also,during the epoxidations the catalysts gradually appeared to precipitateout of solution, most likely due to cleavage of the ester bond betweenthe polypeptide and the solubilising support.

To avoid these difficulties, amino-PEG [H₂N(CH₂CH₂O)_(X)CH₂CH₂NH₂]wasinstead used as the initiator. As amines are generally more nucleophilicthan alcohols or water, trace water present in the sample cannot competeeffectively as an initiator for the leu-NCA; furthermore the resultingamide linkage to the polypeptide is stable under the basic epoxidationconditions and the catalysts do not therefore precipitate from thereaction solution.

Example 2 Synthesis and Testing of Polyleucine on Amino-PEG (1st Batch)

Leu-NCA (164.5 mg, 1.048 mmol) and amino-PEG (140 mg, 0.4179 mmol, 1/25eq) were dried overnight at a temperature of 90° C.(the amino-PEG hadalso been previously dried at 1.1 mbar for 5.5 hr) and stirred togetherin dry THF (1 5 ml) under nitrogen.

IR after 3 days showed much PLL (1654 cm⁻¹) and little NCA. After 7 daysdiethyl ether (250 ml) was added to precipitate the product, which waswashed in a sinter with more diethyl ether (3×100 ml) and dried to yield268 mg (a quantitative yield) of the white solid [H(L-Leu)_(X)NH]₂PEG(X=12.5 by assumption).

Testing of the 1st Batch Amino-PEG-PLL for Homogeneous Epoxidation in(a) THF and (b) CCl₄

60.3 mg of the product was placed in separate vials and stirred in (a)THF and (b) CCl₄ respectively for 1 hour. The solution (a) andsuspension (b) were then filtered through paper to two separate vials(a) and (b) respectively, to which a further 1 ml of the solvent wasadded.

To (a) was added chalcone (50 mg), UHP (35 mg, 1.5eq) and DBU (75 μL, 2eq).

To (b) was added 4M NaOH (2 ml), chalcone (50 mg) and 30% aq H₂O₂ (0.25ml). A further 0.25 ml was added to (b) after 8 hrs.

Time/h % C (a) % ee (a) % C (b) % ee (b) 0.5 31 43 — — 1 50 34 — — 2 7231 — — 3 81 31 13  5 8 96 32 19 13 20 98 29 35 55 50 — — 53 59Testing of the 1st Batch Amino-PEG-PLL for Homogeneous Epoxidation in(c) DME/H₂O, and (d) Toluene/NaOH

To (c) 59.6 mg and (d) 59.5 mg of the polyleucine was added DME/H₂O 1:1(2 ml) and toluene/NaOH 1:1 (2 ml) respectively. After 4 hr stirring,(c) and (d) were filtered through paper to vials, using a further 2 mlof the same solvent mixtures.

To (c) was added chalcone (50 mg) and Na₂CO₃.1.5H₂O₂ (56 mg, 1.5 eq).

To (d) was added chalcone (50 mg) and H₂O₂ (30% aq, 0.5 ml).

Time % C (c) % ee (c) % C (d) % ee (d) 1 h 21 0  0 — 21 h 51 0 19 1 7 d54 0 21 0

Conditions (e), H₂O₂ aq, DBU and THF gave a rapid reaction but no ee.

Example 3 Synthesis and Testing of Polyleucine on Amino-PEG (2nd Batch)

Preparation: Leu-NCA (1.198 g, 20 eq) and amino-PEG (0.6384 g) werestirred in THF (30 ml) for 5 days. IR showed no NCA remained, so theproduct was precipitated with diethyl ether (500 ml), washed with moreether in a sinter and dried, yielding 1.3557 g (90% ) of the product.

Testing: 100 mg of this partially soluble (37% in THF) catalyst wasstirred with UHP (100 mg) in THF (1.8 ml) and DBU (0.2 ml) for 20 min,then filtered to a vial containing chalcone (50 mg) in THF (2 ml).Progress was monitored by HPLC (see over).

Time/h % C % ee 1 56 80 2 62 77 3 63 75  5* 66 73 24  99 55 *a furtheroxidising solution (100 mg UHP/2 ml THF/20 min stir) was filtered inusing another 1 ml THF.

A sample of this catalyst was sent for microanalysis.

Only 6 mg of the catalyst was recovered from a sinter afterprecipitation with ether, this gave an ee of 2-5% when tested exactly asabove with 50 mg chalcone, due to the background reaction. It isbelieved that testing with the substrate and oxidant used on a smallerscale would have seen a higher ee.

Further Work with the 2nd Batch Amino-PEG-PLL

Subsequently it was shown that higher ees could be obtained with thisbatch of catalyst, and an improved recovery procedure utilising membranefilters increased the ee with recycled material.

The 2nd batch amino-PEG-PLL (1.05 g) was stirred in THF (100 ml) andfiltered after 80 min. From the sinter was recovered 345 mg insolublecatalyst. It seems likely that this product is due to trace moisturegetting in the NCA during storage and causing some to polymerise priorto the addition of the amino-PEG initiator. Soluble catalyst 391 mg (37%) was recovered from the filtrate. 104 mg of the soluble and insolublefractions were placed in separate vials with THF (4 ml) and chalcone (50mg). Oxidising solution X (1.4 mL) was then added, and the reactionsmonitored by HPLC. Oxidising solution Y(1.4 ml) was added after 3 h toboth reactions:

Time % C (insol) % ee (insol) % C (sol) % ee (sol) 1 h 33 93 45 92 2 h45 91 58 95 3 h 56 91 66 94 5 h 63 89 77 93 8 d 99 87 99 93

X: UHP (200 mg), DBU (0.4 ml), THF (3.6 ml) stirred for 20 min thenfiltered.

Y: As X except the DBU is replaced by THF (0.4 ml).

Recovery Procedure

The soluble fraction was precipitated with diethyl ether and recoveredusing HPLC membrane filters, which were separately washed carefully withdiethyl ether and water, collected and washed with THF to recover thecatalyst, along with some by-products e.g. epoxychalcone. The THFsolution was dried (MgSO₄), filtered and the solvent removed in vacuo togive 213 mg of solid material containing catalyst (48% max). 1 00 mg ofthis product was tested in exactly the same conditions as before, exceptfor the timing of the addition of the second oxidising solution:

Time % C % ee 1 27 62 4 48 49  5* 58 44 23  96 32 *oxidising solution Yadded.

Example 4 Synthesis and Testing of Polyleucine of Average Chain Lengths5-25 on Amino-PEG

Preparation of the L-Leucine NCA

L-Leucine (15.00 g, 0.1144 mol) was heated to 100° C. under vacuumovernight, suspended in THF (150 ml) and stirred at 45° C. in a 3-neckflask with a reflux condenser fitted, under N₂ pressure. Triphosgene(13.574 g, 0.4 eq) was added via a solid addition tube. After ca 80 min,the reaction had gone clear and the mixture was washed with dry diethylether (1 L) through the filter agent Celite (5 cm deep) in a sinterfunnel (diameter 8 cm porosity 3) to a RB flask.

The solvents were removed in vacuo and the residue redissolved in thesmallest possible volume (ca 80 cm³) of THF. Hexane (1 L) was added toprecipitate out the NCA; the flask was placed in an ice bath for 30 min.The precipitate was filtered off and dried to obtain 8.88 g (49% ) ofL-Leu-NCA as a white crystalline solid.

Polymerisation of the Leu-NCA

Five reactions were set up, with the intended products being[H(L-Leu)_(X)NH]₂PEG with X=5, 10, 15, 20, 25. The Soxhlet extractorwashed amino-PEG was used as the initiator.

5 mer: Initiator 0.5146 g; L-Leu NCA 0.3667 g(10 eq); THF 50 ml

10 mer: Initiator 0.5146 g; L-Leu NCA 0.7333 g(20 eq); THF 50 ml

15 mer: Initiator 0.5146 g; L-Leu NCA 1.100 g(30 eq); THF 50 ml

20 mer: Initiator 0.5146 g; L-Leu NCA 1.4667 g(40 eq); THF 100 ml

25 mer: Initiator 0.5146 g; L-Leu NCA 1.8333 g(50 eq); THF 100 ml

Calculations of equivalents based on microanalysis result; N content ofwashed amino-PEG=1.27% (all N assumed to be available as initiator) andthe NCA MW (157.1679). All reactions had a few activated 4 Å molecularsieves added to the flask and were stirred at RT under N₂.

Workup: After 8 days each reaction was added to ether (500 ml) andwashed into a sinter funnel. The sieves were extracted with tweezers,washing with diethyl ether. Ether (2×200 ml) was used to wash theproduct, then a new flask was placed under the sinter funnel and thesoluble product collected by washing with THF (2×100 ml) followed byremoving the filtrate in vacuo. The mass of the insoluble polyleucineremaining in the sinter was also recorded in each case.

Reaction Yield of soluble polyleucine Yield of insoluble polyleucine 5mer 0.53 g 0.210 g 10mer 0.37 g 0.756 g 15mer 0.49 g 1.137 g 20mer0.45 g 0.942 g 25mer 0.62 g 1.748 gMicroanalysis on these Samples

The soluble samples were submitted for microanalysis, and there are twoways to analyse the data obtained:

(i) If we assume the amino-PEG is fully bifunctionalised, the N contentof 1.27% puts the MW at 2205.

(ii) From the MALDI data (section 13) the average MW is approximately3470. The nitrogen content of 1.27% means that the MW of nitrogen in onepolymer of PEG is on average 44.069, putting the average degree offunctionalization at 3.1462.

Whether method (i) or (ii) is used, we first have to use themicroanalysis data to calculate the proportion of PLL in each sample. Weknow that the percentage of N in the leucine repeat unit is 12.37% andin amino-PEG it is 1.27% , so where the sample N content falls (betweenthese two values) will give the percentage of polyleucine in our sample.The microanalysis results for our catalysts are as follows:

Chain length % N found % C found % H found % PLL in sample  5 4.54, 4.5356.29, 56.30 9.25, 9.27 28.6 10 6.09, 6.10 58.18, 58.24 9.49, 9.47 43.415 7.44, 7.45 58.47, 58.60 9.44, 9.46 55.5 20 7.33, 7.28 58.34, 58.189.45, 9.42 54.3 25 8.04, 8.02 59.24, 59.15 9.44, 9.43 60.9Example of Calculation:% N (PLL)×Proportion PLL (Y)+% N (PEG)×Proportion (PEG)=% N (SAMPLE)×112.378% Y+(1−Y)×1.27%=% N (SAMPLE)$Y = \frac{{\%\quad N\quad({SAMPLE})} - {1.27\%}}{11.108\quad\%}$  Sofor the 5 mer, % N (SAMPLE)=4.535 (av)${{So}\quad Y},{{{proportion}\quad({PLL})} = {\frac{{4.535\%} - {1.27\%}}{11.108\%} = {0.286 = {28.6\%}}}}$Calculation of the Chain Length, Using Method (ii)${{For}\quad 5{mer}},{\frac{{MW}({PLL})}{{{MW}({PEG})} + {M\quad{W({PLL})}}} = 0.286}$ So MW(PLL)=0.286MW(PEG)+0.286MW(PLL)0.714MW(PLL)=0.286MW(PEG)$\frac{1389.94}{3.1462} = {441.79\quad\left( {{MW}\quad{per}\quad{chain}} \right)}$$\frac{441.79}{113.1589\quad\left( {{leu}\quad{repeat}\quad{unit}} \right)} = 3.904$

Intended average chain length Average chain length according to data 53.904 10 7.473 15 12.156 20 11.595 25 15.154Testing of this Series of Catalysts for Epoxidation

5 vials were loaded with amino-PEG-PLL and chalcone as below:

Intended chain length Catalyst/mg PLL mass/mg chalcone used/mg 5 57.316.84 16.84 10 33.6 14.59 14.59 15 77.0 42.80 42.80 20 16.2 8.80 8.80 2596.0 58.42 58.42

UHP (0.5 g) was dissolved in THF(49.5 ml) and DBU (0.5 ml), pre stirredunder N₂ pressure for 20 min then filtered. 0.1 ml/mg chalcone was addedto each reaction after 0 and 4 hr. After 4 days , UHP (570 mg) wasstirred in THF (25 ml), stirred under N₂ for 20 mins and filtered. 0.1ml/mg chalcone of this solution was also added to each reaction.

Chain length 5mer 10mer 15mer 20mer 25mer Time % C/% ee % C/% ee % C/%ee % C/% ee % C/% ee  1 h 39/96.9 39/97.1 34/97.2 36/97.7 26/97.2  2 h38/97.4 39/96.4 33/97.4 37/97.6 27/96.4  4 h* 37/97.2 39/97.8 33/97.635/—   28/96.6 24 h 80/97.5 80/96.6 58/96.4 63/95.0 39/92.8  4 d*81/97.4 77/95.8 59/96.4 68/92.6 42/91.1 10 d 98/96.0 100/96.1  88/87.894/85.4 66/77.9 *extra oxidant added (see above).Titrations on Solubility of UHP in THFEquation:5H₂O₂+2KMnO₄+3H₂SO₄=MnSO₄+K₂SO₄+8H₂O+5O₂ MW 34.012 MW 158.04 98% aq.UHP=H₂NCONH₂.H₂O₂, MW 94.07

Solution 1: UHP (0.500 g) was dissolved in THF (49.5 ml) and DBU (0.5ml), stirred for 20 min at RT and filtered. 36.35 cm³ of this solutiondecolourised 33.4 mg KMnO₄ in H₂O/H₂SO₄, so conc. of H₂O₂ present=0.0145mol.dm⁻³, 100% dissolution of the H₂O₂ from the UHP would be 0.1063mol.dm⁻³, so % dissolution=13.67%.

Solution 2: As above, except 0.550 g UHP used and the mixture stirredfor 40 min. 30.85 cm3 of this solution neutralised 53.1 mg of acidifiedaqueous KMnO₄, so conc. of H₂O₂ present=0.0218 mol.dm⁻³. 100%dissolution here would have been a conc. of 0.1169 mol.dm⁻³, so 18.66%of the H₂O₂ dissolved this time.

Example 5 Synthesis and Testing of Polyleucine of Average Chain Length 3on Amino-PEG (2 Batches)

First 3 mer- Preparation: Leu-NCA (0.2143, 3 eq) and the Soxhletextractor washed amino-PEG (0.5014 g) were stirred in THF (50 ml) underN₂ pressure at RT. IR after 2 days showed no NCA present, so diethylether (200 ml) was added to precipitate the product, which was washed ina sinter with more ether (2×100 ml). THF (4×50 ml) was used to extractthe soluble polymer product, the THF being removed in vacuo to yield[H(L-Leu)₃NH]₂PEG (566.5 mg). Microanalysis: C=54.55, 54.59: H=9.30,9.32: N=2.97, 2.99: Av. chain length=1.77.

Second 3 mer- Preparation: Exactly the same as above, even the same NCAwas used. This had been stored in a dessicator for the weeks betweenthese preparations. For the testing of these Catalysts see Example below“Further Test Results With 5-25 mers”.

Example 6 Synthesis, in Methanol of Polyleucine of Average Chain Length5 on Amino-PEG

Leu-NCA (69.1 mg, 5 eq) and amino-PEG (97.0 mg, dried but not wad in aSoxhlet extractor) were stirred under N₂ in AR methanol containinganhydrous MgSO₄ (1.17 g). IR after 2 days showed no NCA, so the methanolwas removed, the solid product washed in a sinter funnel several timeswith diethyl ether, and the soluble product extracted with THF (28.6mg). For test results see Example below “Adsorbtion of a SolubleCatalyst onto Silica”.

Example 7 Synthesis of Exact Chain Length (1,2,3,4) Polyleucine onAmino-PEG

The scheme below outlines a synthesis of soluble polymer supportedpeptides:

Synthesis of [H(L-Leu)₁NH]₂PEG

Coupling: FmocLeuOH (2.884 g, 2 eq) was dissolved in dry DMF (70 ml),and stirred under N₂ at RT. DIC (1.278 ml, 2 eq) was added, followed byHBTU (3.095 g, 2 eq) and then amino-PEG (4.500 g, 1 eq). The couplingwas monitored by Kaiser testing¹⁰. After 6 days, the solvent was removedin vacuo and the solid product washed in a Soxhlet extractor withrefluxing diethyl ether (ca 10 min/cycle for 3 h) and the solubleproduct extracted with THF (Ca 30 min/cycle, overnight). NMR of theether soluble fractions (mainly excess FmocLeuOH) showed no PEG to bepresent.

Deprotection: The THF soluble product, [Fmoc(L-Leu)l NH]₂PEG wasdissolved in dry DMF (80 ml) and piperidine (20 ml), and stirred underN₂ at RT for 20 h. The solvents were removed in vacuo and the solidproduct washed in a sinter with diethyl ether (×5) to remove thedeprotection by-product, then the soluble product was washed through thesinter with THF, which was subsequently removed in vacuo to leave[H(L-Leu)₁NH]₂PEG (5.09 g, quantitative yield).

Synthesis of [H(L-Leu)₂NH]₂PEG

Coupling: [H(L-Leu)₁NH]₂PEG (4.54 g, 1 eq) was dissolved in dry DMF (60ml), and stirred under N₂ at RT. FmocLeuOH (2.572 g, 2 eq), HBTU (2.760g, 2 eq) and DIC (1.140 ml, 2 eq) were added. A Kaiser test, after 44 h,took 1 min to go blue; the solvent was then removed in vacuo and thesolid product washed several times in a sinter funnel with diethyl ether(3.79 g of ether soluble product was collected). The soluble product(5.25 g, 90% y) was extracted by washing several times with THF.Deprotection: The THF soluble product, [Fmoc(L-Leu)₂NH]₂PEG wasdissolved in dry DMF (80 ml) and piperidine (20 ml), and stirred underN₂ at RT for 20 h. The solvents were removed in vacuo and the solidproduct washed in a sinter with diethyl ether (×5) to remove thedeprotection by-product (0.92 g), then the soluble product was washedthrough the sinter with THF, which was subsequently removed in vacuo toleave [H(L-Leu)₂NH]₂PEG (4.11 g). An NMR spectrum was obtained (section13).

Synthesis of [H(L-Leu)₃NH]₂PEG

Coupling: [H(L-Leu)₂NH]₂PEG (3.59 g, 1 eq) was dissolved in dry DMF (50ml), and stirred under N₂ at RT. FmocLeuOH (1.908 g, 2 eq), HBTU (2.048g, 2 eq) and DIC (0.845 ml, 2 eq) were added. A Kaiser test, after 48 h,took 30 s to go blue, however the solvent was still removed in vacuo atthis time and the solid product washed several times in a sinter funnelwith diethyl ether (1.47 g of ether soluble product was collected). TheTHF soluble product (4.79 g) was extracted by washing several times withTHF, then removing this solvent in vacuo.

Deprotection: The THF soluble product, [Fmoc(L-Leu)₃NH]₂PEG wasdissolved in dry DMF (80 ml) and piperidine (20 ml), and stirred underN₂ at RT for 2 days. The solvents were removed in vacuo and the solidproduct washed in a sinter with diethyl ether (×5), then the solubleportion was washed through the sinter with THF, which was removed invacuo to leave [H(L-Leu)₃NH]₂PEG (4.53 g). It was found that althoughwashing the product with toluene extracted much of the yellowdiscoloration which had accumulated at this stage of the synthesis, PEGwas observed in the NMR spectrum of the residue left by removing thetoluene.

Synthesis of [H(L-Leu)₄NH]₂PEG

Coupling: [H(L-Leu)₃NH]₂PEG (3.24 g, 1 eq) was dissolved in dry DMF (50ml), and stirred under N₂ at RT. FmocLeuOH (1.59 g, 2 eq), HBTU (1.705g, 2 eq) and DIC (0.704 ml, 2 eq) were added. After 48 h the solvent wasremoved in vacuo and the solid product washed several times in a sinterfunnel with diethyl ether. The soluble product (4.26 g) was extracted bywashing several times with THF, then removing this solvent in vacuo.

Deprotection: The THF soluble product, [Fmoc(L-Leu)₄NH]₂PEG wasdissolved in dry DMF (60 ml) and piperidine (15 ml), and stirred underN₂ at RT for 2 days. The solvents were removed in vacuo and the solidproduct washed in a sinter with diethyl ether (×5), then the solubleproduct was washed through the sinter with THF, which was removed invacuo to leave the product [H(L-Leu)₄NH]₂PEG (3.39 g). It was found thatalthough stirring the product with decolourising charcoal extracted onlysome of the yellow coloration (2.25 g of product was recovered afterthis purification attempt).

Testing of the Exact Chain Length Polyleucines in the UHP Conditions

Catalyst (50 mg of 1 mer, 2 mer, 3 mer or 4 mer) and chalcone (25 mg,0.120 mmol) is placed in a vial. UHP (1.5 g) is stirred under N₂ in THFfor 40 min, filtered and titrated: 20 ml of this solution is found todecolourise 116.8 mg of acidified aqueous KMnO₄. 1.8 ml of this solution(1.38 eq of H₂O₂) is then added to each vial, together with DBU (27 uL,1.5 eq) to begin the reactions. After 17 h, UHP (100 mg) and DBU (100ml) are added directly into each reaction.

1mer 2mer 3mer 4mer Time % C/% ee % C/% ee % C/% ee % C/% ee  1 h  3/1 — 19/11  7/9 15 h 20/3 19/4 23/6 13/7 40 h 98/1 96/2 85/3 98/1

Example 8 Further Test Results with the 5, 10, 16. 20 and 25 mers

Using THF/TBME/H₂O₂ Conditions:

Soluble catalyst (Xmg such that 1×10⁻⁵ moles of chains of polyleucinewere present) and chalcone (50 mg) were placed in vials and pre-stirredin THF (1 ml) and DBU (56 uL, 1.5 eq) for 25 min. H₂O₂ in TBME (0.5 ml,1.69 eq) was then added to start the reaction.

To make this solution, 12 ml of 30% aq H₂O₂ was dried for 30 min withMgSO₄ (20.0 g) in TBME (50 ml), then filtered to another conical flaskcontaining MgSO₄ (20.0 g), more TBME (20 ml) was used for thefiltration. This suspension was stirred for 50 min. After filtration,0.5 ml of this solution decolourised 25.7 mg of acidified aqueous KMnO₄.The concentration of H₂O₂ in this solution was shown to be stable withtime, but the solution may not have been properly dry (high rate ofbackground reaction).

Molecular sieves dry H₂O₂ solutions in TBME. A 92% ee was obtained forthe epoxidation of chalcone with a solid polyleucine catalyst, but theconcentration of H₂O₂ decreases rapidly (e.g. from 0.8M to 0.2M in 10min) so this method couldn't be used for adding a solution of oxidant tovarious reactions sequentially. HPLC results are tabulated below:

% C % ee % C % ee % C % ee PLL used Xmg (2 h) (2 h) (4 h) (4 h) (20 h)(20 h) B'GR'ND 0 41 1 56 1 63 0 1st 3mer 13.0 43 3 61 2 94 2 2nd 3mer*13.0 35 4 44 3 70 5  5mer# 15.6 53 23 72 18 100 18 10mer 19.5 53 23 7019 98 14 15mer 24.8 62 29 63 35 96 29 20mer 24.1 51 15 68 12 96 1025mer† 28.2 52 29 59 26 65 27 CLAMPS 43.7 72 43 98 31 100 37 *Nomicroanalysis data on 2nd 3mer, so PLL content was assumed to be thesame as the first. †Used earlier to test out these conditions, with ahigher ee (52% at 49% C and 44% at 90% C). It seems likely that thissolution was drier than that used for the simultaneous tests carried outabove. #Anhydrous TBHP (a solution in decane) gave a very low ee withthe soluble 5mer, though the reaction did proceed.

Example 9 Adsorbtion of a Soluble Catalyst onto Silica¹¹

The 10 mer soluble polyleucine (213 mg) and silica (725 mg) were stirredslowly in THF (6 ml) at RT for 48 h. The resulting silica adsorbedcatalyst was filtered into a sinter funnel, where it was washed with THF(3×10 ml), EtOH (3×10 ml) and THF again (2×10 ml). From the sinter 692mg of silica-bound 10 mer was collected, from the filtrate 147 mg of the10 mer was recovered. Along with the 5 mer of soluble polyleucine grownin methanol, these 2 catalysts were tested in the homogeneous UHP/THFconditions used for testing the 5 mer- 25 mer series.

5 mer (MeOH) grown (silica free)

To Catalyst (11.7 mg) and chalcone (11.7 mg) was added solution X (1.17ml). After 2 h and 23 h was added solution Y (0.29 ml). 10 mer (onsilica, or free from it)

To catalyst (45.7 mg) and chalcone (45.7 mg) was added solution X (4.57mL). After 2 h and 23 h was added solution Y (1.14 ml).

Solution X: UHP (0.5 g) was stirred under N₂ at RT for 20 min in THF(49.5 ml) and DBU (0.5 ml) and filtered.

Solution Y: UHP (2.02 g) was stirred under N₂ at RT for 20 min in THF(50 ml) and filtered.

free 10mer 5mer grown in MeOH 10mer on silica from filtrate Time % C/%ee % C/% ee % C/% ee 1 h 1/63 13/91 39/98 2 h 2/61 27/81 41/98 6 h 6/5842/90 87/97 23 h 7/58 66/87 98/97 48 h 12/47  78/84 100/97 

Example 10 Preparation & Polymerisation of Leucine-NCA

Preparation of Leucine-NCA

Leucine (15 g, 11.44 mmol) was dried under high vacuum overnight at 90°C. and after cooling to 50° C. was suspended in THF (180 ml).Triphosgene (13.51 g, 0.4 eq) previously dissolved in THF (50 ml) wasslowly added over 30 minutes. The mixture was stirred for 1 h afterwhich a clear solution was obtained. After stirring for a further 1 h,the solvent was removed in vacuo and the residue dissolved in a minimumamount of toluene (ca 100 ml). Precipitation of the product wasinitiated by the addition of n-hexane (ca 200 ml). The product wasfiltered, washed twice with n-hexane (2×100 ml) and dried to obtain12.17 g (85% ) of L-leucine-NCA as a white crystalline solid.

Polymerisation of the leucine-NCA

The reaction was set up with the intended product being H(L-Leu),NHPEG-Ywhere Y=MeO and X=15. MeO-PEG-NH₂ was used as initiator.

15 mer initiator 5.00 g; L-Leu-NCA 2.40 g (15 eq); THF 200 ml

The reaction was monitored by IR. After 5 days, only a small amount ofL-Leu-NCA was observed and after 7 days, all the L-Leu-NCA wascompletely consumed.

Workup: After 7 days, the reaction mixture was concentrated to about ⅓its original volume and the solid precipitated by adding diethyl ether(300 ml). The suspension was stirred in an ice bath for 30 minutes andthe resulting solid filtered, washed with ether (2×150 ml) and driedunder vacuum for 2 h to yield 6.5 g of crude material. This wasdissolved in THF (250 ml) and filtered through paper to removedinsoluble residues. The filtrate was concentrated in vacuo and theproduct precipitated by adding ether (200 ml). This was filtered, washedwith ether (2×100 ml) and dried under vacuum for 6 h to give 4.5 g (ca66% ) of H(L-Leu)_(x)NHPEG-Y.

Various modifications and variations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection With specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in chemistry, or related fields are intended to be withinthe scope of the following claims.

References

-   1 For reviews see (a) Ebrahim, S.; Wills, M. Tet rahedron Asymm.    1997, 8, 3163; (b) Pu, L. Tetrahedron Asymm. 1998, 9, 1457; (c)    Porter, M. J.; Roberts, S. M.; Skidmore, J. Bioorg. Med. Chem. 1999,    7, 2145.-   2. Julia, S., Masana, J., Vega, J.; Angew. Chem., Int. Ed. Engl.,    1980, 19, 929.-   3. Bentley, P. A.; Bergeron, S.; Cappi, M. W.; Hibbs, D. E.;    Hursthouse, M. B.; Nugent, T. C.; Pulido, R.; Roberts, S. M.;    Wu, L. E. J. Chem. Soc. Chem. Commun., 1997, 739.-   4. Allen, J. V.; Drauz, K-H.; Flood, R. W.; Skidmore, J. Tet Lett.    1997,40, 5417.-   5. Adger, B. M.; Barkley, J. V.; Bergeron, S.; Cappi, M. W.;    Flowerdew, B. E.; Jackson, M. P.; McCague, R.; Nugent, T. C.;    Roberts, S. M. J. Chem. Soc, Perkin Trans. 1, 1997, 3501.-   6. Cappi, M. W.; Chen, W-P., Flood.; Liao, Y-W., Roberts, S. M.;    Skidmore, J., Smith, J. A.; Williamson, N. M. J. Chem. Soc. Chem.    Commun., 1998, 1159.-   7.Gravert, D. J.; Janda, K. D.; Chem. Rev., 1997, 97, 489.-   8. Bentley, P. A.; Cappi, M. W.; Flood, R. W.; Roberts, S. M.,    Smith, J. A.; Tetrahedron Lett, 1998, 39, 9297.-   9. Volk, M.; Petty, S. A; Org. Lett 2001.-   10. NovaBiochem Catalogue, 1999, pS43 ( For Kaiser test); E. Kaiser,    et.al., Anal. Biochem., 1970, 34, 595.-   11. Geller, T. G.; Final Report, p7.

1. A process for the addition of a nucleophile across an electron poorcarbon-carbon double bond (a Michael addition) comprising contacting ina solvent (i) a nucleophile; (ii) a compound comprising an electron poorcarbon-carbon double bond; and (iii) a catalyst comprising a solublepolymer (SSL) and a polyamino acid (PAA) wherein said catalyst issoluble in the solvent.
 2. A process according to claim 1 wherein thenucleophile is selected from oxygen and sulphur.
 3. A process accordingto claim 2 wherein the nucleophile is oxygen.
 4. A process according toclaim 3 wherein the oxygen nucleophile is provided by a peroxide group.5. A process according to claim 2 wherein the sulphur nucleophile isprovided by a ⁻SPh group.
 6. A process according to claim 1 wherein theelectron withdrawing group is selected from carbonyl, —CN and —NO₂.
 7. Aprocess according to claim 6 wherein the electron withdrawing group andthe carbon-carbon double bond comprise an enone group.
 8. A processaccording to claim 1 wherein the compound comprising the electron poordouble bond is


9. A process according to claim 1 wherein the catalyst is of the formulaor comprises a group of the formula SSL-linker-PAA, wherein linker is alinker bond or an optional linker group.
 10. A process according toclaim 9 wherein the catalyst is of the formula X-SSL-linker-PAA-Y,wherein X and Y are independently selected from OMe and NH₂.
 11. Aprocess according to claim 9 wherein the catalyst is of the formulaX-PAA-linker-SSL-linker-PAA-Y, wherein X and Y are independentlyselected from OMe and NH₂.
 12. A process according to claim 10 wherein Yis NH₂.
 13. A process according to claim 9 wherein the linker bond is anamide bond.
 14. A process according to claim 1 wherein the polyaminoacid is a homopolymer of one, or a copolymer consisting of two or more,amino acids selected from cysteine, glycine, neopentylglycine, alanine,valine, leucine, norleucine, phenylalanine, tyrosine, serine, cystine,threonine, methionine, di-iodotyrosine, thyroxine, dibromotyrosine,tryptophan, proline, hydroxyproline, aspartic acid, glutamic acid,β-hydroxyglutamic acid, ornithine, arginine, lysine and histidine.
 15. Aprocess according to claim 14 wherein the polyamino acid is ahomopolymer of one, or a copolymer consisting of two or more, aminoacids selected from leucine, alanine and neopentylglycine.
 16. A processaccording to claim 1 wherein the soluble polymer is a homopolymer or acopolymer of monomers selected from styrene, vinyl alcohol, ethyleneimine, acrylic acid, methylene oxide, ethylene glycol, propylene oxideand acrylamide.
 17. A process according to claim 1 wherein the solublepolymer is polyethylene glycol (PEG).